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Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4612-4621
By
From the Division of Research Immunology/Bone Marrow Transplantation,
Childrens Hospital Los Angeles, and the Department of Pediatrics,
University of Southern California School of Medicine, Los Angeles, CA;
and the Biotechnology Research Laboratories, Takara Shuzo Co, Ltd,
Otsu, Japan.
Recent reports have indicated that there is poor engraftment from
hematopoietic stem cells (HSC) that have traversed cell cycle ex vivo.
However, inducing cells to cycle in culture is critical to the fields
of ex vivo stem cell expansion and retroviral-mediated gene therapy.
Through the use of a xenograft model, the current data shows that human
hematopoietic stem and progenitor cells can traverse M phase ex vivo,
integrate retroviral vectors, engraft, and sustain long-term
hematopoiesis only if they have had the opportunity to engage their
integrin receptors to fibronectin during the culture period. If
cultured in suspension under the same conditions, transduction is
undetectable and the long-term multilineage regenerative capacity of
the primitive cells is severely diminished.
A RESEARCH GOAL VITAL to the field of
human gene therapy is to identify methods for stable transduction of
pluripotent human hematopoietic stem cells (HSC). Retroviral vectors
derived from the Moloney Murine Leukemia Virus are currently the most effective vehicles to stably integrate new genes into the host cell
chromosomes. The major limitation of retroviral-mediated gene transfer
is the requirement for DNA replication in the target cell.1,2 Hematopoietic stem cells are largely quiescent, and exogenous cytokines are commonly used to prompt cell division. For
gene-modified stem cells to contribute to hematopoiesis throughout the
lifetime of the recipient, it is crucial to define methods for
transduction of HSC that do not initiate pathways of terminal differentiation.
Cell-free supernatant addition to marrow on an irradiated stromal
support layer has been reported to be an effective method for transfer
of genes into human hematopoietic progenitors.3-6 In
addition, our group previously showed that the presence of a human
stromal support layer during transduction was essential to preserve the
ability of human CD34+ cells to sustain durable, long-term
hematopoiesis in immune deficient mice.7 While stromal
support increases levels of gene transduction of hematopoietic cells
and preserves the clonogenic capacity, obtaining sufficient quantities
of autologous stromal cells for clinical trials can be problematic and
requires obtaining a bone marrow aspirate from the patient 2 weeks to 1 month before the stem cell harvest. Therefore, alternate methods of
transduction that are equally effective would be preferable.
The use of the carboxyl (COOH) terminal domain of
fibronectin (FN) to efficiently colocalize retroviral particles and
hematopoietic target cells has been described by Moritz et
al8, Hanenberg et al9, and Moritz
et al.10 Their studies showed efficient transduction of
human long-term culture-initiating cells (LTCIC) and
murine reconstituting cells. In the current studies, we examined transduction of human stem and progenitor cells on the carboxy-terminal domain of FN11 in the bnx/hu xenograft model. The bnx/hu
model of human hematopoiesis allows development of both T lymphocytes and myeloid cells from individual human hematopoietic stem or progenitor cells.7 We used the bnx/hu system to address an issue perhaps more crucial than the transduction level; the survival and maintenance of clonogenic capacity of primitive human
reconstituting cells through a period of ex vivo culture. This factor
is critical for any consideration of gene therapy using ex vivo
manipulated bone marrow cells in fully myeloablated patients to ensure
sustained production of both myeloid and lymphoid compartments from the transplanted cells.
Our previous studies identified two populations of hematopoietic cells
from human bone marrow or mobilized peripheral blood stem cells (PBSC)
that are capable of sustaining multilineage hematopoiesis in
immune-deficient mice. The first population survives ex vivo culture in
suspension with fetal calf serum and the cytokines, interleukin-3
(IL-3), IL-6, and stem cell factor (SCF), but can only sustain
hematopoiesis for 4 to 5 months in the mice.7 The second
population of cells, which we believe to be more primitive, requires
stromal support during ex vivo culture to maintain the ability to
engraft the mice and give rise to hematopoiesis for up to 1 year.7 We ascertained that the late graft failure observed with cells cultured in suspension could be partially, but not completely, ameliorated by inclusion of the cytokine FLT3
ligand (FL),12 which has been shown to stimulate the
survival and/or proliferation of primitive human hematopoietic
cells.13-17 In the current studies, we sought to determine
whether a more crucial factor for maintaining the primitive population
through a 72-hour ex vivo culture was engagement of the integrins very
late antigen-4 (VLA-4) and/or VLA-5 to the FN
carboxy-terminal domain. The effects of transduction of human
CD34+ cells on stromal support versus FN or in suspension
culture (over bovine serum albumin [BSA]-coated plates) were
examined. The transduced cell populations were assessed for the extent
of gene transfer into 14-day colony-forming progenitors and for
survival and transduction of more primitive cells able to sustain human
hematopoiesis in bnx mice for 6 to 12 months.
As we had previously observed, human cells cultured for 72 hours in
suspension culture had a long-term survival defect and were not
recovered in significant levels from any mouse. Human cells cultured 72 hours on stromal support or FN before transplantation had the highest
rates of gene marking and engraftment and gave rise to equivalent
percentages and lineages of mature human hematopoietic cells and
progenitors in the marrow of the mice. Our data indicate that
engagement of the integrins on HSC that bind FN (VLA-4 and VLA-5)
throughout a 72-hour ex vivo culture with serum and cytokines is
necessary to sustain the ability of the cells to mediate long-term hematopoiesis after transplantation.
Preparation of CD34+ cells and stromal monolayers.
Normal human bone marrow cells were obtained from screens used to
filter marrow during harvest of allogeneic donors. Use of these samples
was approved by the Committee on Clinical Investigations at Childrens
Hospital Los Angeles. Cells were incubated in basal bone marrow medium
(BBMM) for 2 hours at 37°C with 5% CO2 to allow depletion of adherent stromal cells before isolation of hematopoietic progenitors. BBMM is Iscove's Modified Dulbecco's Medium (Irvine Scientific, Santa Ana, CA) with 20% heat-inactivated fetal calf serum
(FCS; BioWhittaker, Walkersville, MD), 1% deionized BSA, fraction V,
Sigma, St Louis, MO), 100 U/mL penicillin, 10 µg/mL streptomycin, 2 mmol/L L-glutamine (all from GIBCO, Grand Island, NY),
10-4 mol/L 2-mercaptoethanol, and 10-6 mol/L
hydrocortisone (Sigma). After the stromal depletion step, the
nonadherent hematopoietic cells were collected by vigorous flushing,
and the plastic-adherent fraction was used to generate stromal cell
monolayers as described below. CD34+ progenitors were
isolated by incubation with the monoclonal antibody HPCA-1 (Becton
Dickinson, San Jose, CA), followed by goat antimouse conjugated
immunomagnetic beads (Dynal, Oslo, Norway) as described.18
Transduction of human CD34+ progenitors.
CD34+ cells were transduced in 25-cm2 vent-cap
flasks (Costar) that had been previously coated with BSA, FN, or
stromal cells. FN coating was done by incubating 5 mL of a 50 µg/mL solution of recombinant CH-296 (Retronectin,
TaKaRa, Otsu, Japan) in a 25-cm2 flask for 2 hours at room
temperature. The FN solution was removed and replaced with a 2% BSA
solution (Fraction V; Sigma) to block nonspecific sites for an
additional 30 minutes. BSA coating was performed by incubation of a 2%
solution in 25-cm2 flasks for 2 hours at room temperature.
FN and BSA flasks were rinsed in phosphate-buffered
saline (PBS) and used directly without drying. CD34+ cells
were added to each flask in 5 mL transduction medium and returned to
the incubator for 15 to 30 minutes to reach 37°C and the proper pH
before introduction of virus. Transduction medium was BBMM with 50 U/mL
rH IL-6 (R & D Systems, Minneapolis, MN), 10 ng/mL rH IL-3 (Immunex
Corp, Seattle, WA), and 50 ng/mL SCF (R & D Systems). FLT3 Ligand
(generously donated by DNAX Corp, Palo Alto, CA), was added to
designated flasks at a concentration of 100 ng/mL. For transduction, 5 mL of freshly thawed retroviral supernatant warmed to 37°C was
added to each flask (5 mL Dulbecco's modified Eagle's medium
[DME] with 10% FCS for sham flasks). Supernatant from
the PG13/LN Gibbon Ape Leukemia Virus (GALV) pseudotype packaging cell
line19 was used for all transductions. The PG13/LN
supernatant had a titer of 5 × 106 infectious
virions/mL, assayed on the human colon carcinoma cell line HT29
(American Type Culture Collections [ATCC], Rockville, MD) and was
determined to be free of ecotropic, amphotropic, and GALV recombinant
helper virus by polymerase chain reaction (PCR) for env and
marker rescue assay on 3T3 and HT29 cells (ATCC). After the 72-hour in
vitro transduction period, samples were plated in methylcellulose-based
colony-forming unit (CFU) assays with and without the selective agent
G418 (0.9 mg/mL active, screened lots from GIBCO/BRL, Grand Island, NY)
to assess the extent of gene transfer into 14-day colony-forming
progenitors as described.20 The remainders of each sample
were transplanted into immune-deficient mice.
Mice.
All studies used 6-week old beige/nude/xid homozygous mice
(bg.bg/nu.nu/xid.xid, NIH-3; Taconic Farms, Germantown,
NY) bred at CHLA. Cotransplantation of transduced human
progenitors and stromal cells producing IL-3 was performed as
previously published, with sublethal conditioning performed by
injection of 150 µg/kg 5-fluorouracil (5-FU) 48 to 72 hours before injection of human marrow.18 Mice were killed
6 to 12 months after transplantation with human cells. Bone marrow was
flushed from the tibiae and femurs of each mouse and used for the
assays described below.
Fluorescence-activated cell sorting (FACS) analysis.
Single cell suspensions from the marrow and spleen of cotransplanted
mice were preincubated for 15 minutes on ice with unconjugated mouse
immunoglobulin (MsIgG; Coulter, Hialeah, FL). Directly conjugated antibodies used to identify human-specific cell surface antigens were:
HLE-1 (anti-CD45; Becton Dickinson [BD]), My9-RD1 (anti-CD33, Coulter), Leu-12 (anti-CD19, BD), Leu-3a (anti-CD4, BD), and Leu-2a (anti-CD8, BD). The antimouse CD45-R-PE antibody (Pharmingen, San
Diego, CA) was used to identify murine leukocytes. After a 15-minute
antibody binding period on ice, cells were depleted of red blood cells
by resuspension in Ortho Lysis Buffer (BD), washed, and fixed in 1%
paraformaldehyde. Samples were acquired on a Becton Dickinson FACScan
and analyzed using the Cellquest software package (BD). Ten thousand
events were acquired for each sample. Parallel staining and FACS
analyses were done on normal human and nontransplanted bnx
mouse bone marrow controls to confirm that none of the human-specific
antibodies cross-reacted with murine cells.
Human-specific CFU plating.
To determine the number of clonogenic human hematopoietic progenitors
engrafted within the murine bone marrow, cells harvested from each
mouse were plated in human-specific CFU assay as
described.7,12,18 Before plating, the bnx/hu bone
marrow cells were incubated in BBMM for several hours to remove (by
adherence) murine stromal cells, which secrete murine cytokines and
invalidate the specificity for growth of human hematopoietic colonies
measured in the assay. A total of 5 × 104 and 1 × 105 plastic nonadherent cells from engrafted and
control mice were plated in duplicate in 1 mL of the medium in gridded
culture dishes (Nunc, Naperville, IL), with and without G418 (0.9 mg/mL
active compound; Gemini Bioproducts).
Inverse PCR.
DNA preparation and PCR for the neo gene was performed as described
from total bone marrow and individual T-cell and myeloid clones,
containing 200 to 1,000 cells.18,21,22 DNA from the tissues
of each bnx/hu mouse was isolated by large-scale DNA
preparation using standard methods as described.20,23 For
inverse pCR analysis of neo-positive samples, half of the DNA obtained
from each colony or 50 ng DNA from the tissues was digested with Taq1
restriction enzyme (GIBCO/BRL). A portion of each sample was then
self-ligated and subjected to nested inverse PCR reactions as
described.21,22
Statistical analyses.
The significance of each set of values was assessed using the
two-tailed T-test assuming equal variance with the Excel 5.0 software (Microsoft Corp, Seattle, WA). Average values are listed with
standard deviations, and standard error of the mean was used if all
values were listed in table format.
Transduction of human CD34+ progenitors.
Human bone marrow was enriched for CD34+ progenitors by
immunomagnetic separation. Enriched populations were 90% to 99%
CD34+, as demonstrated by FACS analysis.24 The
CD34+ cells were transduced by addition of supernatant from
the retroviral vector LN, packaged in a GALV pseudotype by the cell
line PG13.19 The LN vector carries the bacterial neomycin
resistance gene25 and imparts resistance to the selective
agent G418. Three supernatant additions were performed at 24-hour
intervals over the course of 72 hours. The cytokines IL-3, IL-6, and
SCF were present in all cultures, with or without inclusion of FLT3
ligand. Transductions were performed in suspension culture (over
BSA-coated plates), on stromal support, or on plates coated with the
recombinant FN fragment CH-296 (Retronectin). A portion of each
transduced sample was plated in 14-day colony-forming assay with or
without the selective agent, G418, to assess the extent of gene
transfer into committed progenitors. The remainder of each sample was
transplanted into a set of bnx mice to allow the subsequent
study of more primitive human hematopoietic cells.
Engraftment of human hematopoietic cells in bnx mice.
After in vitro transduction, each sample was cotransplanted with
primary human stromal cells engineered to produce human IL-3 into
6-week old beige/nude/xid mice, as described.18 To
transplant a series of 12 mice, in three groups, 4 × 106 purified CD34+ cells were initially
inoculated into three separate transduction flasks to transplant four
mice from each flask. The cell numbers transplanted after the in vitro
period were not recounted or normalized, but reflected the input cell
number.
Tissue distribution of vector-marked human cells in bnx mice.
It was possible that the differences in culture conditions during
transduction, before transplantation, could cause variation in homing
and/or sites of subsequent human hematopoiesis. The use of
retroviral marking allows the tracking of a portion of the input cell
population to each organ and can identify differences that might arise
due to the method of ex vivo manipulation of the transplanted cells.
Samples of the bone marrow, blood, and spleen from each long-term
engrafted bnx mouse were therefore analyzed by FACS for the
presence of human CD45+ cells, and organs (liver, lung,
kidney, spleen, and bone marrow) were screened for the presence of LN
provirus by PCR for the neo gene as described.18
Vector-marked cells were most commonly detected in the bone marrow. No
cells bearing neo provirus were recovered from the marrrow of
mice transplanted with human cells transduced in suspension culture
with IL-3, IL-6, and SCF (BSA-FL, Table 3). Addition of FL to the same
transduction condition resulted in the presence of vector-marked cells
in the marrow of two of seven mice (BSA + FL). Nine of the 10 mice that
received human cells transduced in media containing IL-3, IL-6, and SCF
with stromal support harbored cells marked with LN provirus in their bone marrow (stroma-FL, Table 3), and all eight mice transplanted with
human cells transduced in 3/6/S/FL had marked marrow cells (Stroma + FL, Table 3). Nine of 11 mice transplanted with cells cultured on FN
without FL had marked marrow cells, and eight of eight from the FN+FL
group had neo-positive bone marrow. Only two mice from each group
transplanted with cells cultured on stroma or FN had marked human cells
in the spleen at the time of harvest, and no human cells bearing LN
provirus were recovered from the liver, lung, or kidney of any animal
(Table 3). Therefore, the major site for survival of marked human
hematopoietic cells was the murine bone marrow, with few marked cells
recovered from the other tissues after long-term engraftment.
Transduced human colony-forming progenitor recovery from the bone
marrow of bnx mice.
To further assess transduction levels of primitive human cells and
survival of vector-marked human hematopoietic progenitors, human-specific colony-forming assays were plated with and without the
selective agent G418 from the bone marrow of each mouse in the six
groups. In two cases (mice 5D1 and 6F1, Table 3), the total marrow
sample was positive for the neo gene, but no transduced progenitors
were recovered. This data indicates that the neo gene in the total
marrow sample was amplified from mature, marked cells that had lost
clonogenic potential. Mice that had been transplanted with human cells
transduced in the absence of stromal support, FN, or FL had no
long-term engrafted human cells able to form colonies in the presence
or absence of G418 (BSA-FL, Table 3). Only two of the seven mice
transplanted with human CD34+ cells cultured on BSA-coated
plates with FL had G418-resistant human CFU in their marrow (average,
3.9% ± 3.0%, P > .05). In contrast, the majority of the
mice transplanted with cells cultured on stromal support or FN, ± FL, had reclonable, G418-resistant human hematopoietic progenitors in
their marrow. The average percentages of G418R progenitors
in each group were 7.7 ± 2.5 for STRO-FL, 6.5 ± 1.6 for
STRO+FL, 7.9 ± 2.2 for FN-FL, and 12.9 ± 2.9 for FN+FL.
Although all of the groups of mice transplanted with cells transduced
on stromal or FN support had significantly higher levels of transduced, clonogenic progenitors than the BSA-FL group (P < .05), there was no statistical difference in the levels between the
four stromal support or FN groups (P > .05, Fig 2). This data indicates that stromal
and FN support were equivalent in their ability to promote survival and
successful retroviral-mediated transduction of primitive, long-term
engrafting human hematopoietic progenitors.
Clonal integration analysis.
Each marrow sample that had been identified to contain cells marked by
the LN vector (Table 3) was further subjected to inverse PCR to allow
determination of the number of individual, marked human progenitors
contributing to the neo-positive PCR signals. Retroviral
vectors integrate into host cell chromosomes at random sites,
generating unique restriction fragment length polymorphisms. Integrated
vectors can therefore be used as clonal tags to identify all cells
derived from a common progenitor.26-30 The results of the
inverse PCR analyses are summarized in Table 3. Mice that had received
human cells transduced in suspension with IL-3, IL-6, and SCF had no
integrated LN provirus in their marrrow. Only mice #4B1 and 4B3,
transplanted with human CD34+ cells transduced in
suspension with FL, had single vector integrants detectable in their
marrow (Fig 3). Mice that had received
human cells transduced on stromal or fibronectin support ± FL had
oligoclonal marking, with zero to six marked human progenitors
contributing to hematopoiesis (Table 3, Fig 3). The average values in
the latter four groups were not significantly different (P > .05). We conclude that transfer of the neo gene into a limited number of individual human progenitors capable of engrafting bnx mice for
extended periods of time occurred to equivalent levels using either
stromal or FN support during the transduction.
Our previous studies demonstrated that the presence of a stromal
underlayer had dual benefits during ex vivo transduction of long-lived
human CD34+ cells with cell-free retroviral supernatant. In
addition to the enhancement of gene transfer into clonogenic
progenitors, as had been previously shown by Moore et al,3
we discovered that the presence of stroma during ex vivo transduction
also maintained the ability of human progenitors to sustain long-term
hematopoiesis in the bnx/hu system.7,12 We postulated that
engagement of integrins was required to prevent induction of pathways
of terminal differentiation in the cells. Wang et al31
found that primitive hematopoietic cells were rescued from apoptosis by
binding to stromal cells via the VLA-4 and vascular cell adhesion
molecule-1 (VCAM-1) integrins. Chertkov et
al32 had previously reported that murine stem cells
cultured on stromal monolayers had better survival and levels of gene
marking than cells kept on gelatin-coated plates with cytokines. We had
shown that addition of FLT3 ligand to cells cultured in suspension
could partially, but not completely, relace stromal support in
maintaining the capacity of cultured cells to sustain long-term
engraftment.12 Therefore, in the current studies, we
compared culture in suspension with stromal and FN support, in the
presence and absence of FLT3 ligand.
We thank Donald Kohn, Ken Weinberg, Gay Crooks, Craig Jordan, and
Robertson Parkman, as always, for useful discussion. This work would
not have been possible without Sally Worttman, who heads our animal
facility with care, and Renee Traub-Workman and Miriam Figueroa, who
maintain the bnx mouse colonies with great dedication. Pat Snow
provided excellent secretarial assistance.
Submitted June 15, 1998;
accepted August 7, 1998.
Address reprint requests to J.A. Nolta, PhD, Division of
Research Immunology/Bone Marrow Transplantation, Childrens Hospital Los
Angeles, and the Department of Pediatrics, University of Southern
California School of Medicine, 4650 Sunset Blvd, Mailstop #62, Los
Angeles, CA 90027; e-mail: jnolta{at}chla.usc.edu.
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Abstract
Introduction
and chemokines,